Nanoceria for the treatment of oxidative stress

ABSTRACT

A process for making nanoparticles of biocompatible materials is described, wherein an aqueous reaction mixture comprising cerous ion, citric acid and ethylenediaminetetraacetic acid in a predetermined ratio, an oxidant, and water is provided along with temperature conditions to directly form, without isolation, a stable dispersion of cerium oxide nanoparticles. These biocompatible cerium oxide nanoparticles may be used to prevent and/or treat oxidative stress related diseases, such as stroke, relapse/remitting multiple sclerosis, chronic-progressive multiple sclerosis, amyotrophic lateral sclerosis, and ischemic reperfusion injury.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to Provisional Application Ser.No. 61/689,806, NANOCERIA FOR THE TREATMENT OF MULTIPLE SCLEROSIS, filedJun. 13, 2012, Provisional Application Ser. No. 61/690,100, NANOCERIAFOR THE PREVENTION AND TREATMENT OF MULTIPLE SCLEROSIS, filed Jun. 18,2012, Provisional Application Ser. No. 61/795,241, BIOLOGICAL EFFECTS OFNANOCERIA IN A MODEL OF MULTIPLE SCLEROSIS, filed Oct. 12, 2012, andProvisional Application Ser. No. 61/796,639, NANOCERIA FOR THE REDUCTIONOF OXIDATIVE STRESS, filed Nov. 16, 2012, the disclosures of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates in general to improvements in the field ofnanomedicine. In particular, the invention relates to cerium-containingnanoparticles prepared with biocompatible materials, to methods ofpreparing such nanoparticles, and to the use of such nanoparticles toprevent and to treat inflammation and oxidative stress related eventsand diseases.

BACKGROUND OF THE INVENTION

Free radical oxidative stress plays a major role in the pathogenesis ofmany human diseases, and in particular, neurodegenerative diseases.Treatment with antioxidants, which may reduce particular free radicalspecies, therefore, might theoretically prevent tissue damage andimprove both survival and neurological outcome. Free radicals inphysiological environments can often be classified as either a reactiveoxygen species (ROS) or a reactive nitrogen species (RNS). Free radicalsare highly reactive chemical species and readily react with proteins,lipids and nucleic acids at a subcellular level and thereby contributeto the progression of various diseases.

The origin of the use of nanoceria in nanomedicine can be traced to theseminal work of Bailey and Rzigalinski, wherein the application ofultrafine cerium oxide particles to brain cells in culture was observedto greatly enhanced cell survivability, as described by Rzigalinski inNanoparticles and Cell Longevity, Technology in Cancer Research &Treatment 4(6), 651-659 (2005). More particularly, rat brain cellcultures in vitro were shown to survive approximately 3-4 times longerwhen treated with 2-10 nanometer (nm) sized cerium oxide nanoparticlessynthesized by a reverse micelle micro emulsion technique, as reportedby Rzigalinski et al. in U.S. Pat. No. 7,534,453, filed Sep. 4, 2003.Cultured brain cells exposed to a lethal dose of free radicals generatedby hydrogen peroxide or ultraviolet light exposures were affordedconsiderable protection by the cerium oxide nanoparticles. In addition,the cerium oxide nanoparticles were reported to be relatively inert inthe murine body, with low toxicity (e.g. tail vein injections producedno toxic effects). While no in vivo medical benefits were reported,benefits were postulated for treatments with these ceria nanoparticles,including reduced inflammation associated with wounds, implants,arthritis, joint disease, vascular disease, tissue aging, stroke andtraumatic brain injury.

However, a host of problems with these particular nanoceria particleswas subsequently reported by Rzigalinski et al. in WO 2007/002662.Nanoceria produced by this reverse micelle micro emulsion techniquesuffered from several problems: (1) particle size was notwell-controlled within the reported 2-10 nanometer (nm) range, makingvariability between batches high; (2) tailing (carryover contamination)of surfactants, such as sodium bis(ethylhexyl)sulphosuccinate, alsoknown as docusate sodium or (AOT), used in the process into the finalproduct, caused toxic responses; (3) inability to control the amount ofsurfactant tailing posed problems with agglomeration when thesenanoparticles were placed in biological media, resulting in reducedefficacy and deliverability; and (4) instability of the valence state ofcerium (+3/+4) over time. Thus, the cerium oxide nanoparticles producedby the reverse micelle micro emulsion technique were highly variablefrom batch to batch, and showed higher than desired toxicity tomammalian cells.

As an alternative, Rzigalinski et al. in WO 2007/002662 reported thebiological efficacy of nanoceria synthesized by high temperaturetechniques, obtained from at least three commercial sources. These newsources of cerium oxide nanoparticles were reported to provide superiorreproducibility of activity from batch to batch. It was further reportedthat, regardless of source, cerium oxide particles having a small size,narrow size distribution, and low agglomeration rate are mostadvantageous. In regard to size, this disclosure specifically assertsthat in embodiments where particles are taken into the interior ofcells, the preferable size range of particles that are taken into thecell are from about 11 nm to about 50 nm, such as about 20 nm. Inembodiments where particles exert their effects on cells from outsidethe cells, the preferable size range of these extracellular particles isfrom about 11 nm to about 500 nm.

Rzigalinski et al. also report that for delivery, the nanoparticles wereadvantageously in a non-agglomerated form. To accomplish this, theyreported that stock solutions of about 10% by weight could be sonicatedin ultra-high purity water or in normal saline prepared with ultra-highpurity water. However, as others have noted, sonicated aqueousdispersions of nanoceria synthesized by high temperature techniques(e.g. obtained from commercial sources) are highly unstable, and settlerapidly (i.e. within minutes), causing substantial variability inadministering aqueous dispersions of nanoceria derived from thesesources.

Rzigalinski et al. report biological efficacy in relatively simple modelsystems, including in vitro cell cultures, orally fed Drosophilamelanogaster fruit flies, and in mice that were tail vein injected withan apparently less than therapeutic dose (300 nanomoles or about 0.2mg/kg).

Yokel et al. in Nanotoxicology, 2009, 3(3): 234-248, describe anextensive study of the biodistribution and oxidative stress effects of acommercial ceria nanomaterial. In particular, a 5% nanoceria dispersionobtained from Aldrich (#639648) was sonicated for 3 minutes and infusedinto rats at 50, 250 and 750 mg/kg nanoceria dose. The nature of anynanoparticle surface stabilizer(s) was unknown for this material. Thesize of the nanoceria particles was characterized by a variety oftechniques and reported to be on average 31+/−4 nm by dynamic lightscattering. Transmission electron microscopy (TEM) revealed that most ofthe particles were platelets with a bimodal size distribution with peaksat 8 nm and 24 nm, along with some particles ˜100 nm. It was observedthat blood incubated for 1 hour with this form of nanoceria hadagglomerates ranging from ˜200 nm to greater than 1 micron, and thatwhen infused into rats, it was rapidly cleared from the blood (half-lifeof 7.5 minutes). Most of the nanoceria was observed to accumulate in theliver and spleen, while it was not clear that any substantial amount hadpenetrated the blood brain barrier and entered brain tissue cells.

Yokel et al. then sought precise control over the nanoceria surfacecoating (stabilizer) and prepared stable aqueous dispersions ofnanoceria by the direct two-step hydrothermal preparation of Masui etal., J. Mater. Sci. Lett. 21, 489-491 (2002), which included sodiumcitrate as a biocompatible stabilizer. High resolution TEM revealed thatthis form of nanoceria possessed crystalline polyhedral particlemorphology with sharp edges and a narrow size distribution of 4-6 nm.Citrate stabilized dispersions of these 5 nm average size ceriananoparticles were reported to be stable for more than 2 months at aphysiological pH of 7.35 and zeta potential of −53 mV. Thus nosonication prior to administration was required.

Results of an extensive biodistribution and toxicology study of thisform of citrate stabilized nanoceria were reported by Hardas et al.,Toxicological Sciences 116(2), 562-576 (2010). Surprisingly, they reportthat compared with the previously studied ˜30 nm nanoceria (Aldrich(#639648), described above), this smaller nanoceria was more toxic, wasnot seen in the brain, and produced little effect on oxidative stress inthe hippocampus and cerebellum. The results were contrary to thehypothesis that smaller engineered nanomaterial would readily permeatethe blood brain barrier.

While cerium oxide containing nanoparticles can be prepared by a varietyof techniques known in the art, the particles typically require astabilizer to prevent undesirable agglomeration. In regard tobiocompatible nanoceria stabilizers used previously, Masui et al., J.Mater. Sci. Lett. 21, 489-491 (2002) describe a two-step hydrothermalprocess that directly produces stable aqueous dispersions of ceriananoparticles that uses citrate buffer as a stabilizer. However, thisprocess is both time consuming and equipment intensive, requiring twoseparate 24 hours reaction steps in heavy closed-reactors.

Sandford et al., WO 2008/002323 A2, report an aqueous preparationtechnique using a biocompatible stabilizer (acetic acid) that directlyproduces nanoparticle dispersions of cerium dioxide without aprecipitation or isolation step, and without subsequent calcination.Cerous ion is slowly oxidized to ceric ion by nitrate ion, and a stablenon-agglomerated sol of 11 nm crystallite size (and approximately equalgrain size) is obtained when acetic acid is used as a stabilizer.

DiFrancesco et al. in PCT/US2007/077545, METHOD OF PREPARING CERIUMDIOXIDE NANOPARTICLES, filed Sep. 4, 2007, describes the oxidation ofcerous ion by hydrogen peroxide at low pH (<4.5) in the presence ofbiocompatible stabilizers, such as citric acid, lactic acid, tartaricacid, ethylenediaminetetraacetic acid (EDTA), and combinations thereof.Specifically, the stabilizer lactic acid and the combination of lacticacid and EDTA are shown to directly produce stable dispersions ofnanoceria (average particle size in the range of 3-8 nm), without anintermediate particle isolation step.

Karakoti et al. in J. Phys. Chem. C 111, 17232-17240 (2007) report adirect synthesis of nanoceria in mono/polysaccharides by oxidation ofcerous ion in both acidic conditions (by hydrogen peroxide) and basicconditions (by ammonium hydroxide). The specific biocompatiblestabilizers disclosed include glucose and dextran. Individual particlesizes as small as 3-5 nm are disclosed, however, weak agglomerates of10-30 nm result. While the source of the colloidal instability is notdescribed, it is believed that the magnitude of the zeta potential ofthese particles may not have been sufficiently large.

Karakoti et al. in JOM (Journal of the Minerals, Metals & MaterialsSociety) 60(3), 33-37 (2008) comment on the challenge of synthesizingstable dispersions of nanoceria in biologically relevant media, so as tobe compatible with organism physiology, as requiring an understanding ofcolloidal chemistry (zeta potential, particle size, dispersant, pH ofsolution, etc.) so as not to interfere with the reduction/oxidation(redox) ability of the nanoceria that enables the scavenging of freeradicals (reactive oxygen species (ROS) and reactive nitrogen species).Karakoti et al. specifically describe the oxidation of cerium nitrate byhydrogen peroxide at low pH (<3.5) in the absence of any stabilizer, aswell as, in the presence of dextran, ethylene glycol and polyethyleneglycol (PEG) stabilizers. Particle sizes of 3-5 nm are reported,although particle agglomeration to 10-20 nm is also reported.

Kim et al. in Angew. Chem. Int. Ed. 2012, 51, 1-6 report that 3 nmnanoceria synthesized by a reverse micelle method and encapsulated withphospholipid-polyethylene glycol (PEG) can protect against ischemicstroke in rats by reducing brain infarct volume and by scavenging ROS.However, higher doses are not protective, and it is believed that thismay be related to surfactant tailing problems, as noted above, thatplague the reverse micelle synthesis method.

There remains a need for efficient and effective methods and agents formediating and ameliorating damage from free radical oxidative stress. Inaddition, a need remains for further improvements in methods for thedirect preparation (i.e. without a particle isolation step) ofbiocompatible dispersions of cerium-containing nanoparticles, forexample, in higher yield, in a shorter period of time and at highersuspension densities, that are sufficiently small in size, capable ofpenetrating a healthy or unhealthy blood brain barrier, more uniform insize frequency distribution, stable and non-toxic in a wide range ofbiological media, with increased cellular uptake and vascularcirculation time in vivo. Additionally, it would be quite useful toproduce medicaments for the prevention and/or treatment of inflammationand oxidative stress related events, such as ischemic stroke andreperfusion injury, and oxidative stress related diseases, inparticular, central nervous system diseases, such as multiple sclerosisand amyotrophic lateral sclerosis, in mammals, and particularly inhumans.

SUMMARY OF THE INVENTION

In accordance with one aspect of the invention, a method of making adispersion of nanoparticles is provided, comprising: forming a reactionmixture comprising cerous ion, citric acid (CA) andethylenediaminetetraacetic acid (EDTA) in a molar ratio (CA/EDTA)ranging from about 3.0 to about 0.1 (i.e. from about 3:1 to about 1:9),an oxidant, and water; optionally, heating or cooling the reactionmixture, and directly forming, without isolation, a dispersion ofcerium-containing nanoparticles.

In a second aspect of the invention, a process of preventing (i.e.prophylactically treating) an oxidative stress related disease, and inparticular, a central nervous system disease, such as multiple sclerosisor amyotrophic lateral sclerosis, comprising administering, prior to theonset of an oxidative stress related disease, an effective amount of acerium-containing nanoparticle prepared in the presence a mixture ofcitric acid and ethylenediaminetetraacetic acid in a molar ratio rangingfrom about 3.0 to about 0.1, is provided.

In a third aspect of the invention, a process of treating an oxidativestress related event or disease, and in particular, a central nervoussystem disease, such as multiple sclerosis or amyotrophic lateralsclerosis, comprising administering, after onset of a disease or event,an effective amount of cerium-containing nanoparticles prepared in thepresence a mixture of citric acid and ethylenediaminetetraacetic acid ina molar ratio ranging from about 3.0 to about 0.1, is provided.

In a fourth aspect of the invention, a nanoparticle comprising ceriumoxide, citric acid and ethylenediaminetetraacetic acid is provided,wherein the molar ratio of citric acid and ethylenediaminetetraaceticacid added during preparation is in a range of about 3.0 to about 0.1.

In a fifth aspect of the invention, a nanoparticle comprising ceriumoxide, citric acid and ethylenediaminetetraacetic acid, wherein themolar ratio of citric acid to ethylenediaminetetraacetic acid rangesfrom about 3.0 to about 0.1, is provided.

In a sixth aspect of the invention, a pharmaceutical composition for theprevention and/or treatment of an oxidative stress related event ordisease, comprises a cerium oxide nanoparticle, wherein the molar ratioof citric acid and ethylenediaminetetraacetic acid added duringpreparation is in a range of about 3.0 to about 0.1, is provided.

In a seventh aspect of the invention, a pharmaceutical composition forthe prevention and/or treatment of an oxidative stress related disease,comprises a cerium oxide nanoparticle capable of penetrating a mammalianblood brain barrier, is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 contains powder X-ray diffraction (XRD) spectra of CA/EDTA ceriananoparticles along with the line spectrum of CeO2 (Cerianite).

FIG. 2 is a TEM micrograph of dried down CA/EDTA ceria nanoparticles.

FIG. 3 is a high resolution TEM micrograph of dried down CA/EDTA ceriananoparticles.

FIG. 4 is a size class distribution chart of the CA/EDTA ceriananoparticles.

FIG. 5 is a plot of Mean Clinical Score as a function of time for thechronic-progressive model of MS for vehicle control and for CeNPsadministered in the preventative and therapeutic treatment regimens.Drug (CeNPs) treatment dosage was 10 mg/kg.

FIG. 6 is a plot of the Mean Clinical Score as a function of time in thechronic-progressive model of MS for the vehicle control, preventativeand therapeutic treatment regimen. Drug (CeNPs) treatment dosage was 20mg/kg.

FIG. 7 is a chart Clinical Score (AUC) over the disease course for thechronic-progressive model of MS for the vehicle control (cont) and forCeNPs administered by the preventative (prev) and the therapeutic (ther)treatment regimens. Drug (CeNPs) treatment dosage was 20 mg/kg.

FIG. 8 is a chart of the Clinical Severity (AUC) of thechronic-progressive model of MS as a function of CeNPs dosage for thepreventative treatment regimen.

FIG. 9 is a chart of the Clinical Severity (AUC) of thechronic-progressive model of MS as a function of CeNPs dosage for thetherapeutic (3 Day Delay) treatment regimen.

FIG. 10 is a plot of Reduction in Disease Severity as a function oftotal ceria (CeNPs) injected into the chronic-progressive model of MS.

FIG. 11 is a plot of Brain Cerium Content as a function of total ceria(CeNPs) injected into the chronic-progressive model of MS.

FIG. 12 is a plot of Mean Clinical Score as a function of time for thechronic-progressive model of MS for the control, for CeNPs administeredin the preventative and therapeutic treatment regimens, and for dailyfingolimod treatments. Drug (CeNPs) treatment dosage was 30 mg/kg.

FIG. 13 is a chart of Decrease in disease severity during the acutephase (days 0-30) of the chronic-progressive model of MS, relative tocontrols, for Fingolimod, and for CeNPs administered by the preventativeand therapeutic (7 Day Delay) treatment regimens.

FIG. 14 is a chart of Decrease in Disease Severity during the chronicphase (days 31-35) of the chronic-progressive model of MS, relative tocontrols, for Fingolimod and for CeNPs administered by the preventativeand therapeutic (7 Day Delay) treatment regimens.

FIG. 15 is a chart assessing disease severity (AUC) through the entiredisease course for the chronic-progressive model of MS for the Control,for CeNPs administered by the preventative (prev) and therapeutic (7 DayDelay) (ther) regimens, and for the Fingolimod (fing) daily treatmentregimen.

FIG. 16 is a plot of Rotarod Test performance as a function of time forthe chronic-progressive model of MS for the control and for CeNPsadministered by the preventative and therapeutic treatment regimens.Drug (CeNPs) treatment dosage was 20 mg/kg.

FIG. 17 is a chart of Rotarod Test performance for thechronic-progressive model of MS as a function of CeNPs dosageadministered by the preventative treatment regimen.

FIG. 18 is a chart of Rotarod Test performance for thechronic-progressive model of MS as a function of CeNPs dosageadministered by the therapeutic (3 Day Delay) treatment regimen.

FIG. 19 is a plot of Hanging Wire Test performance as a function of timefor the chronic-progressive model of MS for the control and for CeNPsadministered by the preventative and therapeutic treatment regimens.Drug (CeNPs) treatment dosage was 20 mg/kg.

FIG. 20 is a chart of Hanging Wire performance for thechronic-progressive model of MS as a function of CeNPs dosageadministered by the preventative and therapeutic (Delayed) treatmentregimens.

FIG. 21 is a plot of Balance Beam Test performance as a function of timefor the chronic-progressive model of MS for the control and for CeNPsadministered by the preventative and therapeutic treatment regimens.Drug (CeNPs) treatment dosage was 20 mg/kg.

FIG. 22 is a chart of Balance Beam Test performance for thechronic-progressive model of MS as a function of CeNPs dosageadministered by the preventative and therapeutic (Delayed) treatmentregimens.

FIG. 23 is a chart of accumulated total ceria in the brain and spinalcord (sc) and in isolated cerebellum tissues taken from C57BL/6 miceinduced with chronic-progressive MS and administered with vehiclecontrol (cont) or with 20 mg/kg CeNPs in the preventative (prev) andtherapeutic (ther) treatment regimens.

FIG. 24 is a chart of ICP-MS results for accumulation of ceria invarious tissues taken from C57BL/6 mice induced with chronic-progressiveMS and administered with 20 mg/kg CeNPs in the preventative andtherapeutic treatment regimens, and sacrificed on Day 42 post diseaseinduction.

FIG. 25 is a chart of Reactive Oxygen Species Level (Light Intensity) inthe brain (cerebellum sections) during the chronic phase (Day 42) ofchronic-progressive MS for C57BL/6 mice treated with vehicle control,Fingolimod, and Ceria (CeNPs) administered by the preventative (30 mg/kgdosage) treatment regimen.

FIG. 26 is a chart of Reactive Oxygen Species Level (Light Intensity) inthe brain (cerebellum sections) during the chronic phase (Day 42) of thechronic-progressive model of MS expressed as a percentage of the controlfor the Fingolimod and CeNPs administered by the preventative (30 mg/kgdosage) treatment regimens.

FIG. 27 contains fluorescence microscopy images of cerebellum brainslices treated with free radical indicator dye CM-DCFDA taken on Day 42from Ceria (CeNPs) treated (preventative treatment regimen) andUntreated Control mice (pseudo-colored images such that higherfluorescence intensity appears as a warmer (e.g. red/orange, lighterareas) color and lower intensity appears as a cooler (e.g. blue/violet,darker areas) color).

FIG. 28 contains microscopy images of mouse cerebellum brain slicestreated with immunohistochemical stain.

FIG. 29 is a plot of Mean Clinical Score as a function of time for therelapse/remitting model of MS for vehicle control and for CeNPsadministered in the preventative and therapeutic treatment regimens.

FIG. 30 is a plot of Balance Beam Test performance as a function of timefor the relapse/remitting model of MS for the control and for CeNPsadministered by the preventative and therapeutic treatment regimens.

FIG. 31 is a plot of Hanging Wire Test performance as a function of timefor the relapse/remitting model of MS for the control and for CeNPsadministered by the preventative and therapeutic treatment regimens.

FIG. 32 is a plot of Rotorod Test performance as a function of time forthe relapse/remitting model of MS for the control and for CeNPsadministered by the preventative and therapeutic treatment regimens.

FIG. 33 is a chart of Clinical Scores (AUC) over the disease course forthe relapse/remitting model of MS for the Control, Sigma-Aldrich, AlfaAesar (1:14 and 1:9 dilutions) and for CA/EDTA ceria nanoparticles (CNRx87) administered by the therapeutic treatment regimen.

FIG. 34 is a chart of Average Balance Beam Score over the disease coursefor the relapse/remitting model of MS for the Control, Sigma-Aldrich,Alfa Aesar (1:14 and 1:9 dilutions) and for CA/EDTA ceria nanoparticles(CNRx 87) administered by the therapeutic treatment regimen

FIG. 35 is a chart of Average Hanging Wire Test performance over thedisease course for the relapse/remitting model of MS for the Control,Sigma-Aldrich, Alfa Aesar (1:14 and 1:9 dilutions) and for CA/EDTA ceriananoparticles (CNRx 87) administered by the therapeutic treatmentregimen

FIG. 36 is a chart of Average Rotarod Test performance over the diseasecourse for the relapse/remitting model of MS for the Control,Sigma-Aldrich, Alfa Aesar (1:14 and 1:9 dilutions) and for CA/EDTA ceriananoparticles (CNRx 87) administered by the therapeutic treatmentregimen

FIG. 37 is a chart of brain deposition results for the relapse/remittingmodel of MS dosed by the therapeutic treatment regimen with CA/EDTAceria nanoparticles (labeled CNRx) compared to various commerciallyavailable nanoceria (24 mg/kg total dosage).

FIG. 38 is a chart of brain ceria content as a function of timefollowing the final injection of CA/EDTA ceria nanoparticles in therelapse/remitting model of MS (24 mg/kg total dosage).

FIG. 39 is a plot of ceria concentration in the blood plasma over a 24hour period for a 10 mg/kg intravenous (IV) injection and for a 50 mg/kgsubcutaneous injection of CeNPs into rats.

FIG. 40 is a chart of Survival Interval (days) for G93A model ALS micetreated with vehicle control and CeNPs (CNRx 87).

FIG. 41 is a chart of LDH accumulation following myocardialischemia/reperfusion via the Langendorff hanging heart procedure formice treated with vehicle control and with CeNPs (CNRx 87) dosed at 20mg/kg on Days −4, −2 and 0.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that elements not specifically shown or describedmay take various forms well known to those skilled in the art. Theinvention is defined by the claims.

As used herein, the term nanoparticle includes particles having a meandiameter of less than 100 nm. For the purposes of this disclosure,unless otherwise stated, the diameter of a nanoparticle refers to itshydrodynamic diameter, which is the diameter determined by dynamic lightscattering technique and includes molecular adsorbates and theaccompanying solvation shell of the particle. Alternatively, thegeometric particle diameter can be estimated by analysis of transmissionelectron micrographs (TEM).

As used herein, various cerium-containing materials are interchangeablydescribed as “ceria”, “cerium oxide” or “cerium dioxide.” It will beunderstood by one skilled in the chemical arts, that the actual oxidicanions present in these materials may comprise oxide anions or hydroxideanions, or mixtures thereof, such as hydrated oxide phases (e.g.oxyhydroxide). In addition, it is known that compositions of matter maybe comprised of solid solutions of multivalent cations, and are termednon-stoichiometric solids. Thus, for oxide phases comprised of metalcations of multiple oxidation states, it is understood that the totalamount of oxidic anions present will be determined by the specificamounts of the various oxidation states of the metal cations present(e.g. Ce³⁺ and Ce⁴⁺), such that charge neutrality is maintained. Fornon-stoichiometric phases nominally described as metal dioxides, this isembodied in the chemical formula MO_(2-δ) wherein the value of δ (delta)may vary. For cerium oxides, CeO_(2-δ), the value of δ (delta) typicallyranges from about 0.0 to about 0.5, the former denoting cerium (IV)oxide, CeO₂, the latter denoting cerium (III) oxide, CeO_(1.5)(alternatively denoted Ce₂O₃). Alternatively, the value of δ (delta)denotes the amount of oxygen vacancies present relative to cerium (IV)oxide (CeO₂). For each oxygen di-anion vacancy present, two cerous ions(Ce³⁺) are present, to preserve charge neutrality.

In one embodiment of the invention, a process is provided comprising:forming a reaction mixture comprising cerous ion, citric acid,ethylenediaminetetraacetic acid (EDTA), an oxidant, and water;optionally heating or cooling the reaction mixture; and directlyforming, without isolation, a stable dispersion of nanoparticles.

In various embodiments, the molar ratio of citric acid to EDTA in thereaction mixture ranges from about 3:1 to about 1:9; from about 3:1 toabout 2:1; and from about 1.2:1.0 to about 1:9.

In various embodiments, the oxidant includes molecular oxygen or air, orcompounds more oxidizing than molecular oxygen (or an ambient atmosphereof air). In other embodiments, the oxidant has an aqueous half-cellreduction potential greater than −0.13 volts relative to the standardhydrogen electrode. In particular embodiments the oxidant is an alkalimetal or ammonium perchlorate, chlorate, hypochlorite or persulfate;ozone, a peroxide or a combination thereof. In a particular embodiment,a two-electron oxidant, such as hydrogen peroxide, is used. Inparticular embodiments, hydrogen peroxide is present in an amountgreater than one-half the molar amount of cerous ion. In still otherembodiments, the amount of oxidant present varies widely in relation tothe amount of cerium ions or other metal ions present.

In a particular embodiment, molecular oxygen is passed through thereaction mixture.

In particular embodiments, the temperature of the reaction mixture isgreater than or less than ambient temperature. In particularembodiments, the reaction mixture is heated or cooled to temperaturesgreater than or less than ambient temperature. In various embodiments,the reaction mixture is heated or cooled to temperatures greater thanabout 30° C., greater than about 40° C., greater than about 50° C.,greater than about 60° C., greater than about 70° C., greater than about80° C. or greater than about 90° C. In a particular embodiment, thereaction mixture is heated or cooled to a temperature less than theboiling temperature of water.

In various embodiments, the nanoparticles formed are amorphous,semi-crystalline or substantially crystalline, or crystalline. In aparticular embodiment the nanoparticles formed are characterized by acubic fluorite crystal structure. In a particular embodiment, thenanoparticles formed are characterized by a cerium oxide crystalstructure.

As used herein, the terms semi-crystalline and substantially crystallinerefer to nanoparticles that have at least some crystalline structure. Asone of ordinary skill in the art recognizes, accurate characterizationof particles becomes increasingly difficult as the particle size becomessmaller because smaller particles have less detectable long-range order.

In at least one embodiment, the nanoparticles are crystalline and may bemonocrystalline or polycrystalline.

In particular embodiments, the crystallinity of the nanoparticles formedis enhanced by heating of the reaction mixture.

In particular embodiments, the nanoparticles formed are dehydrated ordehydroxylated by heating of the reaction mixture.

In various embodiments, the nanoparticles formed have a hydrodynamicdiameter less than 100 nm, less than 80 nm, less than 60 nm, less than40 nm, less than 20 nm, less than 10 nm, less than 5.0 nm, less thanabout 3 nm or less than about 2.0 nm.

In a particular embodiment, the nanoparticles formed have a geometricdiameter less than the hydrodynamic diameter.

In various embodiments, the nanoparticles formed have a coefficient ofvariation (COV) of the particle size, defined as the standard deviationof the particle size divided by the average particle size, less thanabout 15%, less than about 10%, less than about 5%, or less than about3%.

In a particular embodiment, a nanoparticle comprising cerium isprovided. In other embodiments, nanoparticles comprising a cerium oxide,a cerium hydroxide or a cerium oxyhydroxide are provided.

In a particular embodiment, a nanoparticle comprising citric acid,ethylenediaminetetraacetic acid and a cerium oxide, cerium hydroxide orcerium oxyhydroxide, is provided.

In other embodiments, a nanoparticle having a zeta potential less thanor equal to zero is provided. In particular embodiments, a nanoparticlecomprising cerium oxide, citric acid, ethylenediaminetetraacetic acidand having a zeta potential less than or equal to zero is provided. Inparticular embodiments, a nanoparticle comprising cerium oxide, citricacid, ethylenediaminetetraacetic acid, and having a zeta potential lessthan −10 mV, less than −20 mV, less than −30 mV, less than −40 mV orless than about −50 mV, is provided. In particular embodiments, ananoparticle comprising cerium oxide, citric acid,ethylenediaminetetraacetic acid, and having a zeta potential in therange of −15 mV to −30 mV, is provided.

In particular embodiments, a nanoparticle having a zeta potentialgreater than zero is provided. In particular embodiments, a nanoparticlecomprising cerium, citric acid, ethylenediaminetetraacetic acid, andhaving a zeta potential greater than zero, greater than 10 mV, greaterthan 20 mV, greater than 30 mV, greater than 40 mV or greater than 50mV, is provided.

In various embodiments, the zeta potential of the nanoparticle isaltered by adjusting the pH, the citric acid and/orethylenediaminetetraacetic acid content, or a combination thereof, ofthe nanoparticle dispersion.

In a particular embodiment, the zeta potential of the nanoparticle isaltered by adjusting the citric acid and ethylenediaminetetraacetic acidcontent of the nanoparticle dispersion to less than saturation coverage.

In another embodiment, the zeta potential of the nanoparticle is alteredby adjusting both the pH of the nanoparticle dispersion, and the citricacid and ethylenediaminetetraacetic acid content to less than saturationcoverage.

In various embodiments, the dispersion of cerium-containingnanoparticles contains substantially non-agglomerated nanoparticles,greater than 90 percent non-agglomerated nanoparticles, greater than 95percent non-agglomerated nanoparticles, greater than 98 percentnon-agglomerated nanoparticles, and entirely non-agglomeratednanoparticles.

In a particular embodiment, the non-agglomerated nanoparticles arecrystalline, and are alternatively referred to as single particlecrystallites or individual crystallites.

In a particular embodiment, the nanoparticle dispersion formed is washedto remove excess ions or by-product salts. In various embodiments, thenanoparticle dispersion is washed such that the ionic conductivity isreduced to less than about 15 millisiemens per centimeter (mS/cm), lessthan about 10 mS/cm, less than about 5 mS/cm or less than about 3 mS/cm.In particular embodiments, the nanoparticle dispersion formed is washedby dialysis, diafiltration or centrifugation.

In particular embodiments, the nanoparticle dispersions formed areconcentrated to remove excess solvent or excess water. In particularembodiments, the nanoparticle dispersion is concentrated by dialysis,diafiltration or centrifugation.

In various embodiments, the concentration of nanoparticles in thedispersion is greater than about 0.05 molal, greater than about 0.5molal or greater than about 2.0 molal (approximately 35% solids in agiven dispersion).

In particular embodiments, the size distributions of the nanoparticlesare substantially monomodal. In other embodiments, the nanoparticle sizehas a coefficient of variation (COV) less than about 30%, less thanabout 25%, less than about 20%, less than about 15%, less than about 10%or less than about 5%, where the COV is defined as the standarddeviation divided by the mean.

In particular embodiments, various mixing devices known in the art areemployed to stir, mix, shear or agitate the contents of the reactionmixture. In various embodiments, mixers comprising stir bars, marineblade propellers, pitch blade turbines or flat blade turbines are used.In a particular embodiment, a high shear mixer that forces the reactionmixture to pass through a screen comprising holes ranging in size fromfractions of a millimeter to several millimeters, is employed. Inparticular embodiments, a colloid mill or a Silverson® High Shear Mixeris employed. In particular embodiments, one or more of the reactants isintroduced below the surface of the aqueous reaction mixture. In aparticular embodiment, a reactant is introduced below the surface of theaqueous reaction mixture in close proximity to a mixing device.

In one embodiment of the invention, a process of solvent shifting theaqueous nanoparticle dispersion to a less polar solvent composition bymethods disclosed in commonly assigned US Patent Application Publication2010/0152077, is employed. In a specific embodiment, the nanoparticledispersion is passed through a diafiltration column with an organicdiluent comprising, for example, an alcohol or a glycol ether.

In at least one embodiment, the dispersion of cerium-containingnanoparticles is stable for at least 2 months, such as, for example, atleast 12 months.

Without being bound by any theory, the proposed use of cerium oxides forthe treatment of inflammation and oxidative stress related diseases(e.g. ROS mediated diseases) is based in part upon a belief that ceriumoxides may function as catalytic scavengers of free radicals. Theexistence of and facile inter-conversion of cerium in a mixture of Ce³⁺and Ce⁴⁺ valence states may enable cerium oxides to reduce and/oroxidize free radicals to less harmful species in a catalytic orauto-regenerative manner. Redox reactions may occur on the surface ofcerium oxide nanoparticles (CeNPs) that neutralize tissue-damaging freeradicals. For example, it is believed to be desirable to oxidizesuperoxide anion (O²⁻) to molecular oxygen, to oxidize peroxynitriteanion (ONOO⁻) to physiologically benign species, and to reduce hydroxylradical (.OH) to hydroxide anion. This may in turn enable a greatlyreduced dosing regimen in comparison to, for example, sacrificialantioxidants currently available to treat oxidative stress relateddiseases and events.

In particular embodiments, administered nanoceria particles of theinvention are taken into cells through cell membranes and reside in thecellular cytoplasm or in various cellular organelles, such as thenucleus and mitochondria. In other embodiments, the nanoceria particlesof the invention reside in intravascular or interstitial spaces, whereinthey may reduce oxidative stress and inflammation by eliminating freeradicals or reducing autoimmune responses. In a particular embodiment,the immune system invasion of the central nervous system resulting frombreakdown of the blood-brain barrier (BBB) or blood-cerebrospinal fluidbarrier (BCFB) or blood-ocular barrier (BOB) is modulated by nanoceriaparticles of the invention.

In another embodiment, the nanoceria particles of the invention areparticles capable of crossing a mammalian blood brain barrier. Invarious embodiments, nanoceria particles of the invention cross amammalian blood brain barrier and reside in brain parenchyma tissues asaggregates or agglomerates of a size less than about 100 nm, less thanabout 50 nm, less than about 20 nm, less than about 10 nm, less thanabout 5 nm. In a particular embodiment, nanoceria particles of theinvention cross a mammalian blood brain barrier and reside in brainparenchyma tissues as independent, non-agglomerated nanoparticles of asize less than about 3.5 nm.

In particular embodiments, a pharmaceutical composition comprisingnanoceria particles of the invention are specifically contemplated forprevention and/or treatment of oxidative stress related diseases andevents, such as, but not limited to, Alzheimer's Disease, Parkinson'sDisease, Huntington's Disease, amyotrophic lateral sclerosis (ALS),ataxia, Friedreich's ataxia, autism, obsessive-compulsive disorder,attention deficit hyperactivity disorder, migraine, stroke, traumaticbrain injury, cancer, inflammation, autoimmune disorders, lupus, MS,inflammatory bowel disease, Crohn's Disease, ulcerative colitis,stenosis, restenosis, atherosclerosis, metabolic syndrome, endothelialdysfunction, vasospasms, diabetes, aging, chronic fatigue, coronaryheart disease, cardiac fibrosis, myocardial infarction, hypertension,angina, Prizmetal's angina, ischemia, angioplasty, hypoxia, Keshandisease, glucose-6-phosphate dehydrogenase deficiency, favism, ischemicreperfusion injury, rheumatoid and osteo-arthritis, asthma, chronicobstructive pulmonary disease (e.g. emphysema and bronchitis),allergies, acute respiratory distress syndrome, chronic kidney disease,renal graft, nephritis, ionizing radiation damage, sunburn, dermatitis,melanoma, psoriasis, macular degeneration, retinal degeneration,cataractogenesis, among others.

In particular embodiments, a pharmaceutical composition comprisingnanoceria particles of the invention are specifically contemplated forprevention and/or treatment of oxidative stress related cellularpathologies, such as, but not limited to, mitochondrial dysfunction,lysosome and proteasome dysfunction, oxidation of nucleic acids (e.g.RNA and DNA), tyrosine nitration, loss of phosphorylation mediatedsignaling cascades, initiation of apoptosis, lipid peroxidation anddestruction of the membrane lipid environment.

In at least one embodiment, a pharmaceutical composition comprisingcerium-containing nanoparticles made in accordance with the presentinvention are administered in an effective amount to prophylacticallytreat an oxidative stress related disease. As used herein, the phrase“effective amount” means an amount of a pharmaceutical compositioncomprising sufficient active principle (e.g. cerium-containingnanoparticles) to bring about the desired effect. The pharmaceuticallyeffective amount, as recognized in the art, can be determined throughroutine experimentation.

In at least one embodiment, a pharmaceutical composition comprisingcerium-containing nanoparticles made in accordance with the presentinvention are administered in an effective amount to treat symptoms ofan oxidative stress related disease.

In various embodiments, a pharmaceutical composition comprisingnanoceria particles of the invention is administered to a human or anon-human subject, such as another mammal, including, but not limitedto, a canine, a feline, a bovine, an equine, an ovine, a porcine or arodent. Alternatively, the subject of administration can be an animalsuch as a bird, insect, reptile, amphibian, or any companion oragricultural animal.

In various embodiments, nanoceria particles of the invention areadministered in vivo to a subject by topical, enteral or parenteralmethods, including injections, infusions or implantations. Moreparticularly, it is specifically contemplated to administer nanoceriaparticles of the invention by any of the following routes: auricular(otic), buccal, conjunctival, cutaneous, dental, electro-osmosis,endocervical, endosinusial, endrotracheal, enteral, epidural,extra-amniotic, extracorporeal, hemodialysis, infiltration,interstitial, intra-abdominal, intra-amniotic, intra-arterial,intrabiliary, intrabronchial, intrabursal, intracardiac,intracartilaginous, intracaudal, intracavernous, intracavitary,intracerebral, intracisternal, intracorneal, intracornal-dental,intracoronary, intracorporus cavernosum, intradermal, intradiscal,intraductal, intraduodenal, intradural, intraepidermal, intraesophageal,intragastric, intragingival, intraileal, intralesional, intraluminal,intralymphatic, intramedullary, intrameningeal, intramuscular,intraocular, intraovarian, intrapericardial, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal,intrasynovial, intratendinous, intratesticular, intrathecal,intrathoracic, intratubular, intratumor, intratympanic, intrauterine,intravascular, intravenous, intravenous bolus, intravenous drip,intraventricular, intravesical, intravitreal, iontophoresis, irrigation,laryngeal, nasal, nasogastric, occlusive dressing technique, ophthalmic,oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural,perineural, periodontal, rectal, respiratory (inhalation), retrobulbar,soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual,submucosal, topical, transdermal, transmammary, transmucosal,transplacenta, transtracheal, transtympanic, ureteral, urethral,vaginal, and any other or unassigned route.

In other embodiments, nanoceria particles of the invention are retainedin or on the surface of a medical device or prosthesis, such as acannula, catheter or stent, thereby reducing inflammation locally orsystemically, over either a short or long time period.

In various embodiments, the nanoceria particles of the invention aredelivered in any suitable form known in the art, including, but notlimited to, a suspension, gel, tablet, enteric coated tablet, loadedliposome, powder, suppository, infusible, lozenge, cream, lotion, salve,or inhalant.

In various embodiments, the nanoceria particles of the invention arecombined with other pharmaceutically acceptable substances, such as, butnot limited to, water, salts, buffers, phosphate buffered saline (PBS),sugars, human or bovine serum albumen, lipids, drugs, colorants,flavorants, binders, gums, surfactants, fillers or any excipients knownin the art.

In a particular embodiment, the vehicle comprising the nanoceriaparticles of the invention is sterilized prior to administration.

In other embodiments, a cell or cell culture is contacted with ananoceria particle or particles of the invention. Contact may bepracticed by exposing a cell or cell culture by in vitro or ex vivomethods, wherein the latter method comprises re-introducing the treatedcell or cells into a subject, such as the subject from which the cell orcells were originally obtained. In various embodiments the cell isprokaryotic or eukaryotic in nature. In particular embodiments, thetreated cells are used in the production of proteins used in thepharmaceutical industry, generally known as biologics, such as, but notlimited to, antigens, antibodies and vaccines. In another embodiment,the treated cells are used in a fermentation process.

The invention is further illustrated by the following examples, whichare not intended to limit the invention in any manner.

Experimental Section

Nanoparticle Light Scattering and Size Assessments

A simple qualitative characterization of the particle dispersions wasperformed by assessing the degree of Tyndell scattering exhibited by thedispersions when illuminated by a red laser pen light, relative to theamount of scattering from a sample of the neat solvent. A quantitativeassessment of the particle size of the nanoparticle dispersions wasperformed by dynamic light scattering (DLS) using a Brookhaven 90PlusParticle Size Analyzer (Brookhaven Instruments Corp., Holtzville, N.Y.,U.S.A.) equipped with a quartz cuvette. Reported DLS sizes are thelognormal number weighted parameter.

Nanoparticle Charge Assessment

A quantitative assessment of the nanoparticle charge was made bymeasuring the zeta potential using a Zetasizer Nano ZS from MalvernInstruments.

Preparation of Ceria Nanoparticles with Citric Acid and EDTA

Into a 800 ml glass beaker containing a magnetic stir bar was introduced500 ml of high purity (HP) water. The water was then heated to about 70°C., and therein 2.41 gm of citric acid (CA) and 4.27 gm ofethylenediaminetetraacetic acid, disodium salt (EDTA) were dissolved.Ammonium hydroxide (28-30%) was added to adjust the pH of the solutionto about 8.5. The temperature of the reaction vessel was raised to about80° C., and the magnetic stir bar was replaced with a Silverson® L4RThigh shear mixer operated at 5000 rpm. A 10.0 gm quantity ofCe(NO₃)₃.6(H₂O) was dissolved in 30 ml of HP water, and this solutionwas added slowly to the stirred reaction mixture over several minutes.The reaction pH was maintained at about 8.5 by addition of small amountsof conc. NH₄OH solution. Then a 50 ml solution containing 4.8 ml of 50%H₂O₂ (3.0 molar ratio of H₂O₂ to cerium) was added slowly over severalminutes to the cerous ion, citric acid, EDTA reaction mixture. Thereaction product was covered and then heated for an additional hour,resulting in a clear yellow/orange suspension. After cooling withstirring, the directly formed nanoparticle dispersion was washed bydiafiltration to an ionic conductive of less than about 10 mS/cm, toremove excess salts. The pH of the product dispersion was about 7.2.

The final product dispersion was a clear yellow/orange liquid thatdisplayed a high degree of Tyndall scattering when illuminated with alow intensity LASER beam, indicating it contained well-dispersedcolloidal particles. The final product dispersion was observed to bestable for at least 12 months, with no indication of particleagglomeration or settling. Particle size analysis by dynamic lightscattering on seven replicate preparations yielded an averagehydrodynamic diameter of 3.1 nm with a standard deviation of 0.30 nm(COV of 10%).

Ceria nanoparticles prepared by this method wherein equimolar amounts(50/50) of citric acid and EDTA were added, are referred to hereinvariously as CA/EDTA ceria nanoparticles, CA/EDTA nanoceria, CeNPs,CNRx, or CNRx 87.

The replicate preparations of the CA/EDTA ceria nanoparticles weresubmitted for phase identification and crystallite size analysis bypowder X-ray diffraction (XRD). Sample portions were placed in a Teflonboat, dried under a heat lamp for four hours, and then dried in an ovenfor four hours at 80° C. under vacuum. The resulting solids were lightlyground to form powders. These powders were then front-packed onto glassholders and analyzed by XRD in a N2 dry cell attachment.

Analysis of the XRD spectra of three particular replicate preparationsof the CA/EDTA ceria nanoparticles shown in FIG. 1 indicated that eachsample contained a major crystalline phase iso-structural with CeO₂ (PDF#34-394, cerianite). An average crystallite size of 2.4 nm with astandard deviation of 0.06 nm (COV of 2.5%) in the CeO₂ (220) directionwas determined for the seven replicate samples using the Scherrertechnique.

Moderately high resolution TEM micrographs of dried down CA/EDTA ceriananoparticles (FIG. 2) revealed an ensemble of individual(non-agglomerated) particles with diameters on the order of 2-3 nm.Higher resolution TEM micrographs of dried down CA/EDTA ceriananoparticles (FIG. 3) revealed individual arrays of atoms in selectednanoparticles. A size class distribution was determined from the TEMmicrographs, as shown in FIG. 4.

Zeta potential measurements showed an average charge of −23 mV for theseaqueous dispersions of replicate preparations of CA/EDTA ceriananoparticles.

The preparation of CA/EDTA ceria nanoparticles described above wasrepeated except that the molar ratio of citric acid and EDTA stabilizerswas adjusted to 100/0, 80/20, 70/30, 60/40, 40/60, 30/70, 20/80 and0/100, while maintaining a constant total molar amount of stabilizer.Stable dispersions of cerium oxide nanoparticles with substantiallysimilar physical characteristics (particle size and zeta potential)resulted, as shown in Table 1 below.

Evaluation of Cerium Oxide Nanoparticles in Various Oxidative StressRelated Diseases Ischemic Stroke

Mouse Hippocampal Brain Slice Model of Ischemic Stroke

The ability of nanoceria to reduce oxidative stress was evaluated in amodification of the in vitro mouse hippocampal brain slice model ofischemia described by Estevez, A Y; et al., Neuroprotective mechanismsof cerium oxide nanoparticles in a mouse hippocampal brain slice modelof ischemia, Free Radic. Biol. Med. (2011)51(6):1155-63(doi:10.1016/j.radbiomed.2011.06.006).

Adult (2-5 months of age) CD1 mice were sacrificed via rapiddecapitation and their brains quickly removed and placed in a chilledcholine-based slicing solution containing 24 mM choline bicarbonate, 135mM choline chloride, 1 mM kynurenic acid, 0.5 mM CaCl₂, 1.4 mM Na₂PO₄,10 mM glucose, 1 mM KCl, and 20 mM MgCl₂ (315 mOsm). Transversehippocampal slices, 400 μm thick, were cut along a rostral-to-caudalaxis (−1.2 to −2.8 mm Bregma) using a Leica VT1200 Vibratome (LeicaMicrosystems, Wetzlar, Germany) and allowed to recover for 1 hr in acontrol artificial cerebral spinal fluid (aCSF) containing 124 mM NaCl,3 mM KCl, 2.4 mM CaCl₂, 1.3 mM MgSO₄, 1.24 mM K₃PO₄, 26 mM NaHCO₃, 10 mMglucose and bubbled with 5% CO₂, 95% O₂ gas (pH 7.4, 300 mOsm).Hippocampal slices were placed in a culture dish and stored in a NuAirehumidified incubator (NuAire, Plymouth, Minn., USA) at 37° C. with 5%CO₂ for up to 48 hr.

Oxidative stress from ischemia was induced by placing the brain slicesin hypoglycemic, acidic and hypoxic aCSF (glucose and pH were lowered to2 mM and 6.8, respectively, and the solution was bubbled with 84% N₂,15% CO₂, and 1% O₂) at 37° C. for 30 min. Sucrose was added to maintainthe osmolarity of the solution at about 295 mOsm. Aqueous dispersions ofcerium oxide nanoparticles prepared as described supra were administeredin matched dosage in a delivery volume of 1 μg per 1 ml aCSF or medium(equivalent to 5.8 μM) at the onset of the ischemic event, and remainedin the medium throughout the remainder of the experiment. Control slicesreceived an equal volume of vehicle control. Various delivery vehicleswere used with similar success for the cerium oxide nanoparticlesprepared as described herein, including distilled water alone, salinesolution, Na-citrate solution, PBS, and combinations thereof.

After exposure to 30 minutes of oxidative stress (ischemic conditions),the living brain slices (test and control) were incubated for 24 hr inorganotypic culture by placing them in a 35 mm culture dish containingculture medium and Millipore inserts (Millipore, Billerica, Mass., USA).Culture medium contained 50% minimum essential medium (HycloneScientific, Logan Utah, USA), 25% horse serum, 25% Hank's balanced saltsolution (supplemented with 28 mM glucose, 20 mM HEPES and 4 mM NaHCO₃),50 U/ml penicillin, and 50 μl/ml streptomycin, pH 7.2.

The extent of cell death was measured 24 hours after the oxidativeinjury using fluorescence imaging techniques. Each set of brain slicesstudied in the test condition (i.e. administered with cerium oxidenanoparticles) was matched with a similar set of control brain slicestreated identically in every way except for administration of vehiclealone. Thus on each study day, two sets of anatomically matched brainslices taken from age-matched and sex-matched littermates were subjectedto either the test condition (administered with cerium oxidenanoparticles) or control (vehicle alone). During fluorescence imagingmeasurements, the light intensity, duration of image capture, and timingof image collection were identical for the test condition and vehiclecontrol brain slices. Results were expressed as the ratio of thefluorescence in the test condition to the fluorescence in the matchedcontrol slice imaged at the same time point in the experimentalsequence.

At 24 hours post oxidative injury, paired (control and test) brainslices were incubated for 20 min in culture medium containing 0.81 μMvital exclusion dye SYTOX® Green (Invitrogen, Carlsbad, Calif., USA)and, subsequently, washed for 15-20 min in culture medium to removeunincorporated dye. SYTOX® Green is a fluorescent dye that binds to DNAand RNA. However, it is excluded from the cell nucleus by the cellmembrane in intact, viable cells. Therefore, it acts as a vital dye andstains only those dead and dying cells in which the cell membrane hasbecome permeable so that the dye has access to the cell interior. Afterstaining and washing, brain slices were transferred to the stage of aNikon TE 2000-U (Nikon Instruments, Melville, N.Y., USA) microscopeequipped with epifluorescence attachments and a 150-W xenon light source(Optiquip, Highland Mills, N.Y., USA). Control aCSF solution was loadedinto 60-ml syringes, equilibrated with 95% O₂/5% CO₂, and heated to 37°C. using a servo-controlled syringe heater block, stage heater, andin-line perfusion heater (Warner Instruments, Hamden, Conn., USA). Thebrain sections were continuously perfused with warmed, 95% O₂/5% CO₂equilibrated aCSF at a rate of 1 ml per minute. After 5 min, images ofthe hippocampal formation of each control and test brain slice werecollected using a 4× Plan Flour objective (Nikon Instruments) underidentical conditions (i.e. light intensity, exposure time, cameraacquisition parameters). SYTOX® Green fluorescence was measured bybriefly (620 ms) exciting the tissue at 480±40 nm, filtering the emittedfluorescence (535±50 nm) from the probe using a 505 nm, long-pass,dichroic mirror (Chroma technology, Bennington, Vt., USA), intensifying,and measuring with a cooled CCD gain EM camera (Hamamatsu CCD EM C9100;Bridgewater, N.J., USA). The digital images were acquired and processedwith Compix SimplePCI 6.5 software (C Imaging Systems, CranberryTownship, Pa., USA).

The light intensity resulting from the SYTOX® Green loading reflectedthe number of dead or dying cells within the calculated area. Thelight-intensity measurements were performed automatically using theCompix SimplePCI 6.5 software, thereby eliminating experimenter bias inselecting the regions of interest.

Reduction in cell death is reported as the ratio of the light intensityof SYTOX® Green fluorescence from the cornu ammonis fields (orienslayer, stratum radiatum and lacunosum moleculare) for the test condition(i.e. nanoceria treated) to the control (untreated) for anatomicallymatched hippocampal sections taken from age-matched and sex-matchedlittermate brains sliced and exposed to ischemic oxidative stress on thesame day, and fluorescence imaged 24 hr after the ischemic insult.

Cerium oxide nanoparticles prepared with biocompatible stabilizerscomprising citric acid, EDTA and combinations thereof, were evaluated inthe Mouse Hippocampal Brain Slice Model of Ischemic Stroke using atreatment concentration of 5.8 μM. Results for the reduction in celldeath (percent reduction relative to control), commonly referred to assparing, as a function of citric acid to EDTA molar ratio are given inTable 1 below.

TABLE 1 Sparing Synergy Particle Size Sparing Results Actual- CA/EDTAXRD DLS Actual Predictive Predictive Actual/ Ratio (nm) (nm) (%) (%) (%)Predictive 100/0  2.0 7.8 15.5 15.5 0 1 80/20 2.4 3.4 6.0 12.8 −6.8 0.570/30 2.3 3.8 21.6 11.4 10.2 1.9 60/40 2.4 2.6 11.3 10.0 1.3 1.1 50/502.4 3.1 30.3 8.65 21.65 3.5 40/60 2.5 2.9 26.3 7.3 19.0 3.6 30/70 2.53.0 23.0 5.9 17.1 3.9 20/80 2.4 3.5 6.9 4.5 2.4 1.5  0/100 2.1 2.4 1.81.8 0 1

Treatment with cerium oxide nanoparticles prepared with citric acidalone as a stabilizer (100/0) reduced cell death (sparing) by about 16%,whereas treatment with cerium oxide nanoparticles prepared with EDTAalone as a stabilizer (0/100) had little effect on cell death (1.8%reduction). Further reduction in cell death, alternative termed anincrease in sparing, is a desirable feature of a pharmaceuticalcomposition or medicament. Treatment with cerium oxide nanoparticlesprepared with a combination of citric acid and EDTA in a molar ratioranging from 70/30 to 20/80 resulted in surprising increases in sparingthat substantially exceeded the simple linear predictive additive sumbased on the effects of each stabilizer used alone. For example, thegreatest sparing (about 30%) was seen for the equimolar (50/50) ratio ofcitric acid to EDTA, whereas the simple linear prediction fornanoparticles prepared with this combination of stabilizers is theaverage of a 15.5% sparing for citric acid alone and a 1.8% sparing forEDTA alone, which is only an 8.65% sparing. Thus a surprising andunexpected synergy between the combination of citric acid and EDTAstabilizers has been discovered, wherein the actual sparing forequimolar (50/50) citric acid and EDTA is about 3.5 times larger thanthe simple linear prediction.

In general, a simple linear (additive) model for the Predictive sparingpercent for a given ratio of citric acid to EDTA, is given by theexpression:[Fraction of CA]*[Sparing % of CA]+[Fraction of EDTA]*[Sparing % ofEDTA]wherein the fraction of a given stabilizer is the molar fraction of thetotal stabilizer present. For the results shown in Table 1, Sparing % ofCA is 15.5%, and Sparing % of EDTA is 1.8%. The values of thisexpression (Predictive sparing percent) are tabulated above in Table 1in the column headed by Sparing Results and Predictive (%).

In general, the synergistic increase in sparing can be embodied in twodistinct parameters. The difference between the Actual and Predictivesparing amounts (Actual−Predictive) embodies the synergy on an absolutebasis, for which a positive value represents unexpected additionalsparing (inventive result) and a negative value represent less than theexpected amount of sparing (i.e. a negative interaction or interferencebetween the stabilizers). Alternatively, the ratio of Actual toPredictive (Actual/Predictive) embodies the synergy on a relative basis,for which a value greater than one represents the relative amount ofadditional unexpected sparing (inventive result), and a value less thanone represents the relative amount of sparing less than the Predictiveexpected amount due to a negative interaction or interference betweenthe stabilizers (comparative result).

Examination of these parameters in the Sparing Synergy columns in Table1 reveals, once more, that treatment with cerium oxide nanoparticlesprepared with a combination of citric acid and EDTA in a molar ratioranging from 70/30 to 20/80 resulted in a synergistic increase inabsolute sparing (the value of (Actual−Predictive) is positive) alongwith a synergistic increase in relative sparing (the value of(Actual/Predictive) is greater than one). The greatest amount ofabsolute synergistic sparing increase occurs for the treatment ratio ofcitric acid to EDTA of 50/50, for which an additional 21.65% of sparingis unexpectedly observed. The greatest amount of relative synergistsparing increase occurs for the treatment ratio of citric acid to EDTAof 30/70, for which the Actual sparing is 3.9 times greater than thePredictive.

In contrast, a negative interaction or interference is observed for thetreatment with cerium oxide nanoparticles prepared with a combination ofcitric acid and EDTA in a molar ratio of 80/20, for which the absoluteActual sparing was 6.8% less than the Predictive, or, alternatively, therelative Actual sparing was only one-half (0.5 times) that of thePredictive.

Thus, in summary, it has been discovered that treatment with ceriumoxide nanoparticles prepared with molar ratios of citric acid to EDTA ina range of about 3.0 to about 0.1 resulted in a synergistic increase insparing, whereas treatment with cerium oxide nanoparticles prepared witha molar ratio of citric acid to EDTA of 4.0 resulted in an interferenceleading to less than the expected sparing.

Multiple Sclerosis

Multiple sclerosis (MS) is a disease of the central nervous system (CNS)that affects more than 2 million people worldwide. MS has long beenconsidered an immune mediated inflammatory disease leading, in part, tothe degeneration of the myelin sheath surrounding nerve cells and,ultimately, neuronal cell death due to oxidative stress. The most commoncourse of the disease, termed relapse/remitting, is characterized byclearly defined attacks of worsening neurological and motor function,followed by periods of relative quiet (remission) with no new signs ofdisease activity. A less common course of the disease is termedchronic-progressive MS and is characterized by a steady progression ofclinical neurological damage, without remission after initial MSsymptoms. While only about 20% of patients are initially diagnosed withchronic-progressive MS, about half of those initially diagnosed withrelapse/remitting MS will progress to the chronic-progressing form withthe passage of each decade.

Chronic-Progressive Multiple Sclerosis

Murine EAE Model of MS

Many of the pathological features of the onset of MS are modeled by themurine experimental autoimmune encephalomyelitis (EAE) model, wherein aninflammatory disorder is induced by immunization with myelin antigens.The EAE model is characterized by blood-brain-barrier (BBB) breakdown,perivascular infiltration of immune cells, microglia activation, anddemyelination. The EAE model has been critical in the development ofcurrent therapies used in the treatment of MS.

SJL-EAE mice were purchased from Jackson Laboratories (C57BL/6) andtreated with vehicle or vehicle plus CA/EDTA ceria nanoparticles. TheCA/EDTA ceria nanoparticles mixed in PBS/50 mM sodium citrate salinewere administered to experimental animals by IV tail vein injectioneither before (preventative model) or after (therapeutic model) diseaseinduction and then were given maintenance doses of differentconcentrations. In one experiment a subset of mice were treated dailywith the immunomodulatory drug fingolimod (Cayman Chemical, Ann Arbor,Mich., USA) at 2 μg/L in the drinking water. The various treatmentregimens (dosing regimens) are described in detail in Table 4 below.

TABLE 4 Maintenance doses: Day 7 Administration Day before Induction Day3 post- and weekly Regimen* induction day induction thereafterPreventative 15 mg/kg 15 mg/kg 10, 20, or 10, 20, or 30 CeNPs CeNPs 30mg/kg mg/kg CeNPs CeNPs Therapeutic: — — 10, 20, or 10, 20, or 30 3 DayDelay 30 mg/kg mg/kg CeNPs CeNPs Therapeutic: — — — 30 mg/kg 7 Day DelayCeNPs or Fingolimod**

The mice were induced with experimental autoimmune encephalomyelitis(EAE), i.e. chronic-progressive multiple sclerosis-like symptoms, asfollows: a 0.1 ml intravenous (IV) tail injection of 200 μg myelinoligodendrocyte (MOG₃₅₋₅₅) protein peptide (Genscript) dissolved inphosphate buffered saline (PBS) mixed with an equal volume of completeFreund's adjuvant, was followed by an 0.1 ml intraperitoneal injectionof 200 ng pertussis toxin in PBS was delivered on Days 0 and 2.

Disease progression was scored daily using a Clinical Scoring Testdescribed below, along with the three Motor Behavior Tests designed toevaluate cerebellar function (Balance Beam), forelimb strength (HangingWire), and hindlimb strength (Rotarod).

Clinical Scoring Test

Disease progression of multiple sclerosis type symptoms in the EAE micewas scored daily using a clinical scale adapted from Selvaraj et al.(2008), as shown in the Table 2 below.

TABLE 2 Disease Score Symptoms 0 Normal movement; no paralysis 0.5 Limptail, tail drags when the mouse walks. Mouse can, however, curl tailwhen lifted 1.0 Full tail paralysis; tail drags when the mouse walks.Mouse cannot curl tail when lifted 2.0 Partial limb paralysis; limptail; mouse walks with a clumsy (wobbly) gait; no complete paralysis ofany limbs 2.5 Partial limb paralysis; limp tail; mouse cannot walk,limbs can still move when mouse is lifted 3.0 One hind limb fullyparalyzed; limp tail; mouse drags hind legs, but can still move around3.5 Both hind limbs fully paralyzed; limp tail, mouse drags hind legs,but can still move around and eat 4.0 Both hind limbs paralyzed, onefront limb paralyzed; limp tail, movement severely impaired; mousesacrificedMotor Behavior TestsHanging Wire Test

A hanging wire task was used to assess grip strength. For this task,mice were placed in an open-top Plexiglas box with a steel wire gridfloor. The box was turned upside down 60 cm above the counter top, andlatency to fall was measured.

Rotarod Test

A rotarod apparatus (Med Associates, St. Albans, Vt.) was used to assessmainly hind limb motor coordination and endurance. Mice were placed ontoa drum rotating at 28 rpm and latency to fall from the drum (300seconds, maximum) was measured.

Balance Beam Test

For this task, mice were placed on the illuminated end of an elevatedwooden beam and given up to 60 s to reach the goal box. Balance and gaitquality were scored using a 5 point scale (5=normal gait to 0=falls offbeam immediately). Gait quality was further rated according to the scaledescribed below.

TABLE 3 0 Falls off beam 1 Clings to beam; DOES NOT move with prodding 2Clings to beam for max time; DOES move with prodding: SCOOCHES 2.5Clings to beam for max time; DOES move with prodding: WALKS 3 Alternatesclinging and moving: SCOOCHES; DOES NOT walk entire beam in allowed time3.5 Alternates clinging and moving: WALKS; DOES NOT walk entire beam inallowed time 4 Paces beam but DOES NOT reach goal box in allowed time ORalternates clinging and moving: WALKS; DOES walk entire beam in allowedtime 5 Traverses entire beam without difficulty within time allowed

CA/EDTA ceria nanoparticles decreased (improved) Clinical Scoringresults for both the Preventative and Therapeutic dosing designs areshown for the 10 mg/kg dosage in FIG. 5, and for the 20 mg/kg dosage inFIG. 6. As a measure of cumulative disease severity, the area under thecurve (AUC) of Mean Clinical Score vs. Post-Induction Day (see FIG. 7),was calculated for each animal dosed at the 20 mg/kg level. CA/EDTAceria nanoparticles decreased (improved) Clinical Severity in adose-dependent manner for the Preventative treatment regime (FIG. 8) andthe Therapeutic 3 Day Delay treatment regime (FIG. 9). An overall viewof the reduction in disease severity as a function of total ceriainjected is shown in FIG. 10.

Tissue accumulation of ceria: a subset of mice was euthanized byisoflurane overdose and transcardially perfused with PBS. Harvestedtissues were frozen and analyzed for cerium by inductively coupledplasma mass spectrometry (ICP-MS). Brain cerium content as a function oftotal ceria injected is shown in FIG. 11.

The results of FIGS. 10-11 suggest that particle penetrance into the CNScorrelates well with dose delivered and is not saturated at the range ofdoses tested.

Comparison of CA/EDTA ceria nanoparticles dosed at the 30 mg/kg level tothe immunomodulatory drug Fingolimod is shown in results of FIGS. 12-15.All treatment groups significantly reduced disease severity relative tocontrols during both the A) acute (FIG. 13) and B) chronic phases (days31-35) of the disease (p<0.05) (FIG. 14). The Fingolimod andPreventative treatments were significantly more effective that theTherapeutic (7 Day Delay) treatment during the acute phase. All groupswere equally effective during the chronic phase of the disease (Days31-35).

In addition, treatment with the CA/EDTA ceria nanoparticles improved themotor behavior performance of the mice. Daily group average motorbehavior performance for mice receiving the 20 mg/kg dosage of theCA/EDTA ceria nanoparticles is shown for the Rotorod test (FIG. 16) andHanging Wire Test (FIG. 19), wherein a longer latent time to fallrelative to the control indicates improved motor performance. Dailygroup average Balance Beam performance for the 20 mg/kg dosage is shownin FIG. 21, wherein a higher score relative to the control indicatesimproved motor performance. Motor behavior performance continued toimprove with increasing dosage over the ranges studied, as shown for theRotarod Test for both the Preventative (FIG. 17) and Therapeutic 3 DayDelay (FIG. 18) treatment regimes, and for the Hanging Wire Test (FIG.20) and the Balance Beam Test (FIG. 22) for all doses and treatmentregimes. Results of cerium content analysis by inductively coupledplasma mass spectrometry (ICP-MS) of organs and brain sections isolatedfrom mice treated with CA/EDTA nanoceria are shown in FIGS. 23-24,indicating that cerium accumulated highest in the cerebellum for boththe preventative and therapeutic treatment regimens.

Reactive oxygen species (ROS) levels were studied in brain slicesprepared from the cerebellum of mice that were induced to developed EAE(chronic-progress symptoms of MS), the slices having been prepared 1week after the final CA/EDTA nanoceria injection (n=12 mice). ROS levelswere measured using the fluorescent probe CM-DFCDA (Invitrogen), usingmethods described in Estevez, A Y; et al., Neuroprotective mechanisms ofcerium oxide nanoparticles in a mouse hippocampal brain slice model ofischemia, Free Radic. Biol. Med. (2011)51(6):1155-63(doi:10.1016/j.radbiomed.2011.06.006).

Intracellular ROS levels decreased significantly in brain slices fromCA/EDTA nanoceria treated mice compared to control and to fingolimodtreated animals, when tested 7 days after last drug treatment (FIGS.25-26).

Broad and uniform distribution of nanoceria into the brain parenchymatissues of a living mammal, when imaged at a micron or submicronresolution, has not been previously reported. To this end, the broaddistribution of CA/EDTA nanoceria particles throughout the mouse braintissue is indicated by the diffuse and uniform nature of the decreasedROS fluorescence (CM-DFCDA) levels evident in cerebellar slices takenfrom a CA/EDTA nanoceria treated and untreated paired control (FIG. 27).In particular, it was noted that the distribution of fluorescence in thenanoceria treated slice (FIG. 27) does not correspond to thedistribution of cerebellar microvasculature depicted at a similar scaleof magnification in FIG. 28, suggesting that the CA/EDTA nanoceriaparticles are not limited to the microvascular vessels or trapped in theBlood Brain Barrier cells, but are broadly distributed throughout thecerebellar tissue. These observations are consistent with thepenetration of CA/EDTA nanoceria particles through the compromised bloodbrain barrier of EAE mice induced with chronic-progressive multiplesclerosis, and that the particles were widely dispersed in the braintissues.

Relapse/Remitting Multiple Sclerosis

Murine EAE Model of Multiple Sclerosis

Many of the pathological features of the onset of MS are modeled by themurine experimental autoimmune encephalomyelitis (EAE) model, wherein aninflammatory disorder is induced by immunization with myelin antigens.The EAE model is characterized by blood-brain-barrier (BBB) breakdown,perivascular infiltration of immune cells, microglia activation, anddemyelination. The EAE model has been critical in the development ofcurrent therapies used in the treatment of MS.

Female SJL-EAE mice were treated with vehicle, vehicle plus CA/EDTAceria nanoparticles or vehicle plus commercial nanoceria obtained fromSigma-Aldrich or Alfa Aesar. Commercially obtained nanoceria wasdispersed in vehicle with sonication just prior to use. In thePreventative dosing design, the mice were IV tail vein injected with 10mg/kg of the CA/EDTA ceria nanoparticles on the day prior to diseaseinduction and on the day of disease induction, followed by injections of6 mg/kg of the CA/EDTA ceria nanoparticles on Days 3, 7, 14 and 21 postdisease induction. The Therapeutic dosing design was similar except thatthe first two injections (prior to and day of disease induction) wereeliminated. The CA/EDTA ceria nanoparticles were mixed in PBS/50 mMsodium citrate saline vehicle prior to administration.

The mice were induced with experimental autoimmune encephalomyelitis(EAE), i.e. relapse/remitting multiple sclerosis-like symptoms, asfollows: a 0.1 ml intravenous tail injection of 200 μg myelin basicprotein peptide (PLP139-151) dissolved in phosphate buffered saline(PBS) mixed with an equal volume of complete Freund's adjuvant, wasfollowed by an 0.1 ml intraperitoneal injections of 200 ng pertussistoxin in PBS on Day 0 and Day 2.

Following disease induction, mice developed the first episode ofparalysis 11-14 days (peaked at 14 days) after immunization and, similarto most human MS patients, they fully or almost fully recovered fromthis first wave of paralysis by about Day 20.

Testing included daily clinical scoring along with the three motorbehavior tests designed to evaluate cerebellar function (Balance Beam),forelimb strength (Hanging Wire), and hindlimb strength (Rotarod), asdescribed previously.

In regard to onset of the disease, a substantial delay (improvement) wasseen in the following tests: Clinical Scoring results for both thePreventative and Therapeutic dosing designs (FIG. 29), Balance Beamresults for the Preventative dosing design (FIG. 30), and in the HangingWire results for both the Preventative and Therapeutic dosing designs(FIG. 31).

A statistical summary of the quantitative (average) effects of theCA/EDTA ceria nanoparticles administered by the Preventative andTherapeutic dosing designs compared to vehicle controls is tabulated inTable 6. Statistically significant improvements were shown for ClinicalScoring and each of the motor behavior tests for both Preventative andTherapeutic dosing designs, except for the case of the Preventativedosing design for the Rotarod test.

TABLE 6 Hanging Wire Rotorod Max Max Latency to PLP Model Peak ClinicalScores Latency to Fall Fall Balance Beam Score Control (n = 20) X 1.7 ±0.2 SE X 33.8 ± 10 SE   X 63.3 ± 17 SE X 1.8 ± 0.4 SE vs X 0.9 ± 0.3 SE  X 27.1 ± 5.6 SE X 158.7 ± 39 SE   X 4.1 ± 0.58 SE Preventative (n = 8)p = 0.048 p = 0.799 p = 0.001* p = 0.005* Control (n = 20) X 1.7 ± 0.2SE X 33.8 ± 10 SE   X 63.3 ± 17 SE X 1.8 ± 0.4 SE vs   X 0.9 ± 0.25 SE X100.8 ± 16 SD  X 146.6 ± 37 SE   X 3.1 ± 0.63 SE Therapeutic (n = 12)  p= 0.038*  p = 0.003* p = 0.001* p = 0.005*

Comparison of the average Clinical Scores (AUC) over the disease coursefor the relapse/remitting model of MS indicates that relative to theControl, only the CA/EDTA ceria nanoparticles (CNRx 87) ameliorate thedisease (FIG. 33). Results for Sigma-Aldrich and Alfa Aesar (1:14 and1:9 dilutions) comparisons are either worse or no different from theControl.

Comparison of the average Balance Beam Score over the disease course forthe relapse/remitting model of MS indicates that the CA/EDTA ceriananoparticles (CNRx 87) performed the best, whereas, with the exceptionof the Alfa Aesar 1:9 dilution, comparisons were either worse or nodifferent from the Control (FIG. 34).

Comparison of the average Hanging Wire Test results over the diseasecourse for the relapse/remitting model of MS indicates that relative tothe Control, only the CA/EDTA ceria nanoparticles (CNRx 87) amelioratethe disease by increasing the mean latency time to fall (FIG. 35).Results for Sigma-Aldrich and Alfa Aesar (1:14 and 1:9 dilutions)comparisons are either worse or no different from the Control.

Comparison of the average Rotarod Test results over the disease coursefor the relapse/remitting model of MS indicates that relative to theControl, only the CA/EDTA ceria nanoparticles (CNRx 87) ameliorate thedisease by increasing the mean latency time to fall (FIG. 36). Resultsfor Sigma-Aldrich and Alfa Aesar (1:14 and 1:9 dilutions) comparisonsare worse than the Control.

Comparison of Cerium Brain Levels

Using the Therapeutic dosing design, EAE-induced mice (n=12) receivedtail vein injections of ceria dispersions (24 mg/kg total dosage)comprising the CA/EDTA ceria nanoparticles or a commercially availablenanoceria (i.e. obtained from Sigma-Aldrich and Alfa Aesar). Twenty fourhours after the last injection, the brains and other organs wereharvested and concentration of ceria in these organs was determinedusing inductively coupled plasma mass spectroscopy (ICP-MS).

Brain deposition results shown in FIG. 37 indicate that cerium was belowthe detection limit for Sigma-Aldrich nanoceria, whereas the depositionin the brain of CNRx nanoceria embodiment of the invention is about 4times greater than that of the Alfa Aesar materials.

In a separate biodistribution study, four adult SJL mice between theages of 1-3 months possessing the experimental autoimmuneencephalomyelitis (EAE) characteristic, were tail vein injected with 52mg of CA/EDTA nanoceria per kg of mouse body mass (52 mg/kg dosage) insaline at three time points: Day 0, Day 3 and Day 7. In addition, two ofthese mice were induced to develop multiple sclerosis (MS) like symptoms(experimental autoimmune encephalomyelitis) by injection of proteolipidprotein (PLP) on Day 0, and demonstrated peak, MS-like symptoms by Day7. The other two mice were not induced to develop MS-like symptoms, butwere simply injected with saline as a vehicle control. On Day 8 (24hours after the last injection of nanoceria) each of the four animalswas sacrificed; and their heart, kidney, liver, lung, spleen, brain andspinal cord organs removed, frozen and submitted for cerium contentanalysis.

The organs were analyzed for bulk cerium content using inductivelycoupled plasma mass spectrometry (ICP-MS) by the following procedures. A0.1-0.5 g tissue sample of each of the organs was digested with 1 ml ofoptima HNO₃ in a 15 ml polypropylene tube, and heated to 105° C. for 30minutes in a microwave digestion oven. The sample was allowed to cool,100 μl of H₂O₂ added and the sample diluted to 10 ml final volume withdeionized water. These digested samples were analyzed for bulk ceriumcontent by ICP-MS (7500cx, Agilent, Santa Clara, Calif.) operated innormal mode. The instrument was calibrated with NIST-traceable primarystandards and a second source standard was used as a calibration check.

Table 5 shown below contains the bulk cerium content results for thefour mice (labeled Mouse 1-4) that constitute the biodistribution studydescribed herein as embodiments of the invention. In addition, theresults of earlier whole animal (rodent) biodistribution studies ofintravenously administered nanoceria reported Yokel et al.Nanotoxicology 3(3), 234-248 (2009) (data taken from Table I therein),and Hardas et al. Toxicological Sciences 116(2), 562-576 (2010) (datataken for 20-h termination from Table 2 therein), are included forcomparison.

TABLE 5 Dosage Induced Bulk Cerium Content (mg/kg) Study (mg/kg) MSBrain Heart Kidney Liver Lung Spleen Yokel 50 1 — — 610 — 2828 et al.Hardas 100 0.6 — — 1007 — 2885 et al. Mouse 1 52 Yes 28 767 3798 300281316 17762 Mouse 2 52 Yes 42 1304 6985 31609 1419 17090 Mouse 3 52 No 701325 3343 26141 1807 18814 Mouse 4 52 No 59 1241 4431 25567 6303 21655

Comparison of the bulk cerium content between the studies describedherein (Mouse 1-4) and the earlier studies done at comparable or higherdosage (Yokel et al. and Hardas et al.) indicates that about 30-100times more cerium is associated with the brain, about 25-50 times morecerium is associated with the liver, and about 7 times more cerium isassociated with the spleen as a result of injecting the aqueousdispersion of 2.5 nm diameter CA/EDTA ceria nanoparticles describedherein as an embodiment of the invention. In addition, it is noted thatthe surprisingly large increase in the amounts of cerium associated withthe various organs was observed in both healthy mice (Mouse 3-4) thatpossess a fully intact BBB, as well as in the mice with induced MS-likesymptoms (Mouse 1-2) that are expected to have a substantiallycompromised BBB.

It is noted that differences among in the biodistribution protocolsemployed in the studies described herein (Mouse 1-4) and the earlierstudies done at comparable or higher dosage (Yokel et al. and Hardas etal.) were, in general, quite small in comparison to the large increasesin cerium associated with the various target organs as a result of thisembodiment of the invention. Specifically, Yokel et al. used a 50 mg/kgdose and terminated the animals 20 hours after the final injection.Hardas et al. used a 100 mg/kg dose and also terminated the animals 20hours after the final injection, and the inventors herein used a 52mg/kg dose and terminated the animals 24 hours after the finalinjection.

Bio-Persistence Studies

At different time points (1-21 days) from the last CA/EDTA ceriananoparticle injection (24 mg/kg total dosage), brains of mice inducedwith the relapse/remitting form of EAE-(n=22) were harvested and theconcentration of cerium determined using ICP-MS. Significant levels ofceria were detectable up until at least 3 weeks after the last injection(FIG. 38).

From studies done in rats, following a single 10 mg/kg intravenousinjection or a single 50 mg/kg subcutaneous injection, measurements ofcerium content in the blood of the rats indicate that CA/EDTA ceriananoparticles were cleared quickly from the blood plasma (FIG. 39).

Toxicity Studies

No genotoxicity was observed for the CA/EDAT ceria nanoparticleembodiment when evaluated by the GreenScreen assay of Gentronix Ltd.(UK).

No phospholipidosis toxicity was observed for the CA/EDAT ceriananoparticle embodiment when evaluated by the Phospholipidosis (PLD)assay of Gentronix Ltd. (UK).

No potassium channel interference was observed for the CA/EDAT ceriananoparticle embodiment when evaluated by the hERG-450 assay ofGentronix Ltd. (UK).

Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is progressive, fatal, motor neurondisease caused by the degeneration of upper and lower neurons located inthe ventral horn of the spinal cord and the cortical neurons thatprovide their efferent input. The condition is often referred to as LouGehrig's disease, after the baseball player who was diagnosed with thedisease in 1939.

While the cause of ALS is not known, the discovery that familial ALS isrelated to mutations in the gene that produces Cu/Zn superoxidedismutase enzyme (SOD1), a powerful antioxidant, suggests thataccumulation of free radical may be involved. However, mice lacking theSOD1 gene do not customarily develop familial ALS, rather they exhibitan increase in age-related muscle atrophy (sarcopenia).

SOD1^(G93A) mice obtained from Jackson Laboratory (Bar Harbor, Me., USA;strain B6SJL-TgSOD1^(G93A)) underwent weekly clinical and motor behaviortesting (described above) and were randomized into treatment groups atdisease onset. One group of mice received saline vehicle controlinjections alone, whereas the nanoceria treated animals were given tailvein injections of CA/EDTA ceria nanoparticles of 16 mg/kg either onceor twice per week.

Male G93A mice receiving the nanoceria treatment displayed verysubstantial improvements in all motor skill tests (Hanging Wire, BalanceBeam and Rotarod). An extension in lifespan relative to the control,shown in FIG. 40, was also exhibited by the male G93A mice receiving theCA/EDTA ceria nanoparticle treatment.

Ischemic Reperfusion Injury

Reperfusion injury refers to the tissue damage that occurs when bloodsupply returns to the tissue after a period of ischemia. The absence ofoxygen and nutrients from the blood during the ischemic period creates acondition wherein the restoration of circulation results in inflammationand oxidative damage through the induction of oxidative stress ratherthan the restoration of normal metabolic function.

The inflammatory response is believed to partially mediate the damage ofreperfusion injury. White blood cells carried to the area by newlyreturning blood may release a variety of inflammatory factors, includinginterleukins and free radicals.

In a demonstration of murine cardiac ischemic reperfusion injury, micewere injected with vehicle or CA/EDTA ceria nanoparticles at a 20 mg/kgdosage on Days −4 and −2 via the jugular vein. On Day 0 hearts wereexcised and perfused on a Langendorff system. Necrotic cell death wasmonitored by lactate dehydrogenase (LDH) assay, following 25 min ofglobal no-flow ischemia and a 45 min reperfusion. FIG. 41 shows animprovement in the form of a reduction in LDH accumulation for theCA/EDTA ceria nanoparticle treatment relative to the vehicle control.Assessment of cardiac infarct size also suggested a protective effectwas provided by the 20 mg/kg dose of CA/EDTA ceria nanoparticles.

While the invention has been described by reference to various specificembodiments, it should be understood that numerous changes may be madewithin the spirit and scope of the inventive concepts described.Accordingly, it is intended that the invention not be limited to thedescribed embodiments, but will have the full scope defined by theclaims

What is claimed:
 1. A method of making a dispersion of nanoparticles,comprising: forming a reaction mixture comprising cerous ion, citricacid and ethylenediaminetetraacetic acid in a molar ratio ranging fromabout 3.0 to about 0.1, an oxidant, and water; thereby directly formingin the reaction mixture, without isolation of the nanoparticles, adispersion of cerium-containing nanoparticles.
 2. The method of claim 1,wherein said cerium-containing nanoparticles are substantiallycrystalline.
 3. The method of claim 2, wherein said nanoparticles arecharacterized by a cubic fluorite crystal structure.
 4. The method ofclaim 1, further comprising heating or cooling the reaction mixture tomaintain a reaction temperature less than the boiling temperature ofwater.
 5. The method of claim 1, wherein said oxidant comprises air,molecular oxygen or hydrogen peroxide.
 6. The method of claim 1, whereinsaid cerium-containing nanoparticles are substantially non-agglomerated.7. The method of claim 6, wherein greater than about 95 percent of saidcerium-containing nanoparticles are non-agglomerated.
 8. The method ofclaim 1, wherein said dispersion of cerium-containing nanoparticles hasa zeta-potential ranging from about −15 mV to about −30 mV.
 9. Themethod of claim 1, wherein said dispersion is stable for 2 to 12 months.